DNA vaccines offer many advantages over more traditional approaches to immunization, but the levels of immunity induced by this new approach tend to be rather low. In several studies, protection against viral challenge has been conferred by DNA immunization, but levels of induced immunity were low or undetectable by standard in vitro methods. This proposal focuses on improving the immunogenicity of DNA vaccination, by optimizing several criteria (aims 1-3), and by specifically enhancing immunogenicity by co- administration of immunomodulatory molecules (aim 4). The specific aims are: l. Evaluate different delivery vehicles for DNA. Efficiency of DNA delivery will be assessed when delivered "naked', complexed with cationic lipid. or enclosed in liposomes. The evaluations will employ both marker genes, and immunogenic viral sequences, and both in vitro and in vivo experiments will be undertaken. 2. Evaluate different routes of inoculation. DNA will be administered by several routes, and its expression and immunogenicity determined. Additionally, the laboratory has access to a "gene gun", which allows transdermal DNA delivery. The efficiency of this process will be compared to simple inoculations. 3. Evaluate different promoters. In concert with aims 1 & 2, I will attempt to optimize DNA expression and immunogenicity by using different transcriptional promoters. Three promoters will be employed; one to give general expression, one to direct transcription to muscle, and one to direct transcription to monocyte/macrophages. 4.Improve the immunogenicity of current DNA vaccines by co-expression of immunomodulatory molecules. Enhancement of immunogenicity will be attempted by co-inoculation of immunogenic DNA sequences and immunomodulatory molecules. Two classes of immunomodulator will be used; adhesion molecules, important in the close apposition of, and signaling between, T cells and antigen presenting cells: and cytokines, which may enhance T cell responses. 5.Evaluate the relative efficiency of DNA vaccines in generation of MHC class I-restricted immune responses, compared to generation of MHC class Il-restricted responses. By their nature, DNA vaccines may favor antigen presentation by the MHC class I path way, and disfavor presentation by MHC class II; this may lead to skewing in favor of CTL responses (and against antibody responses). Experiments will be carried out to address this concern. These experiments will be carried out using the LCMV model system. We have extensively characterized the immune response to this agent. and have identified sequences which confer immunity when employed as vaccines. DNA immunization in this system confers protection, though at a level lower than that conferred by more traditional vaccination. Thus we are ideally placed to optimize this DNA vaccine-induced antiviral protective immunity.